Method for Pathogen Identification

ABSTRACT

The present invention relates to a method for detecting a pathogen in cellular lysate by measuring pathogen-specific enzyme activity. The method comprises contacting the cellular lysate with a substrate the pathogen of interest recognizes and modifies, and obtaining a measurable, recordable, signal. The method may comprise detection of SARS-CoV viruses using the activity of SARS PLpro enzyme in tongue scrape lysate as a readout.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. patent application Ser.No. 17/380,862 entitled “Method for Pathogen Identification,” filed Jul.20, 2021, which application is incorporated by reference in itsentirety. The present application also incorporates by reference U.S.provisional patent application Ser. No. 63/053,964, entitled “Method forIdentifying Pathogens for Measurement Thereof,” filed on Jul. 20, 2020.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under contract numberSP4701-21-P-0021. The U.S. government has certain rights in theinvention.

SEQUENCE LISTING

The present application contains a sequence listing file,Pathogen_ID.xml created on May 13, 2023, 9 KB in size, which has beensubmitted electronically in XML format as part of this application, andwhich is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION Field of the Invention (Technical Field)

Embodiments of the present invention relate to a method for determiningenzymatic activity in a sample and a sample preparation protocol. Theinvention is relevant to the field of diagnostics; biological andenzymological research; and pharmacology and medicine.

DESCRIPTION OF RELATED ART

Coronaviruses (CoVs) belong to the subfamily of Coronavirinae within thefamily Coronaviridae and the order Nidovirales. Coronaviruses areenveloped viruses with a single stranded, non-segmented and positive RNAof 27 to 31 kb. The family of Coronaviridae consists of four genera:Alphacoronavirus (Alpha-CoV), Betacoronavirus (Beta-CoV),Gammacoronavirus (Gamma-CoV) and Deltacoronavirus (Delta-CoV). Most ofthe mammalian CoVs belong to Alpha- and Beta-CoV, whereas the avian andthe cetacean CoVs are in the Gamma-CoV.

Coronaviruses enter human host cell through specific attachment toreceptors on host cells. SARS-CoV-1, SARS-CoV-2 and NL63 coronavirusesenter cells by binding to the specific host cell receptor ACE2, which ishighly expressed on epithelial cells of the gastrointestinal tract,lung, and cells of the oral cavity, such as the tongue. In contrast, theMiddle East respiratory coronavirus (MERS) enters cells via the DPP4receptor, 229E via CD13, HKU1 and 0043 via sialic acid. SARS viruses mayenter cells through other receptors, such as CD147 or others not yetidentified.

Three global epidemics of human coronaviruses have so far emerged inthis century. In 2002, infections with severe acute respiratory syndromecoronavirus (SARS-CoV-1) were reported in China. Ten years later theMiddle East respiratory syndrome coronavirus (MERS-CoV) appeared inSaudi Arabia and spread worldwide. In December 2019, a novelcoronavirus—severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)—was identified in Wuhan, China. Current studies indicatethat this coronavirus is similar to SARS-CoV-1. The main symptoms ofthese coronavirus infections are similar to influenza-like complaintsand include fever, headache, malaise, shivering and diarrhea. SARS-CoV-2also infects the lower respiratory tract (bronchial tubes and the lungs)where it can trigger an inflammatory response as the body producesantibodies and T cells to fight the virus. Around 15% of peopleexperience an overreaction of their body's immune system, called acytokine storm. This can severely damage the lungs, kidney and heart andcause clot formation, causing some people to become severely ill or die.

With a fatality rate of ˜2.3%, SARS-CoV-2 is much less fatal thanSARS-CoV-1 (9.5%) or MERS (34.4%). However, because initial symptoms ofCOVID are benign, the virus spreads more easily among asymptomaticcarriers before they experience symptoms.

Coronaviruses (CoVs) are also documented in a wide range of plant andanimal species, including terrestrial and aquatic, domestic and wild.The animal viruses are known to cause mainly gastrointestinal andrespiratory diseases with different severity levels. In certain cases,CoV infections are responsible for huge economic losses. Coronavirusesaffecting livestock and other animals include bovine coronavirus(cattle), bulbaline coronavirus (buffalo), Middle East respiratorysyndrome coronavirus (MERS; dromedaries), equine coronavirus, rabbitcoronavirus, porcine hemagglutinating encephalomyelitis virus (PHEV),transmissible gastroenteritis virus (porcine TGEV), porcine respiratorycoronavirus (PRCV), swine acute diarrhea syndrome coronavirus(SADS-CoV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), infectious bronchitis virus (IBV; chicken),dolphine bottlenose whale (BdCoV), beluga whale (BWCoV), and harbor sealcoronavirus. Other animals that harbor coronaviruses are cats, dogs,mink, tigers, gorillas, and others.

Enzymes are proteins that catalyze chemical or biological reactions bylowering the activation energy of a chemical and/or biological reaction.Thousands of different enzymes exist. The molecules upon which enzymesmay act are called substrates, and the enzyme converts the substratesinto different molecules known as products. Enzymes specificallyrecognize their substrates and form an enzyme-substrate complex. Bindingbrings the substrate into a favorable conformation for a reaction tooccur. The enzymes can thus accelerate up to millions of times thechemical reactions. Like any catalyst, enzymes are neither consumed inchemical reactions, nor do they alter the equilibrium of a reaction.

Enzymes differ from most other catalysts by their specificity for thesubstrates they modify. Enzyme activity can be affected by othermolecules: inhibitors are molecules that decrease enzyme activity andactivators are molecules that increase activity. Many therapeutic drugsand poisons are enzyme inhibitors.

Proteolytic enzymes that cleave a protein or peptide substrate arecalled proteases. Proteases are essential for the biological function ofviruses, eukaryotes, and prokaryotes. Proteolytic enzymes hydrolyzeamides and esters to produce peptides or single amino acids. Proteaseactivity can be measured using naturally occurring substrates or, forexample, using a synthetic peptide substrate with an amino acid sequencethe protease of interest recognizes. The peptide can be labeled with areporter molecule, such as a fluorescent probe or a chromophore. In somecases, the protease activity can be measured using a labeled singleamino acid. The protease activity is measured by the ability of theprotease to cleave the bond between an amino acid and the reporterlabel.

Papain-like proteases are multifunctional enzymes that are essential toviral replication. After a coronavirus enters a cell, it releases itsRNA and hijacks the host replication machinery to translate its RNA intotwo polyproteins, pp1a and pp1ab. The polyproteins are cleaved into 16non-structural proteins (nsps) by the SARS main protease (3CLPro) andeither one or two viral papain-like proteases. Human coronavirusesSARS-CoV-1, SARS-CoV-2, and MERS contain only one papain-like protease(PLpro), whereas HCoV-HKUI, HCoV-0C43, HCoV-NL63 and HCoV-229E containtwo protease domains, PLP1 and PLP2, that process the polyprotein. ThePLpro substrate binding site recognizes three cleavage sequences in thepolyprotein pp1 that share the highly conserved consensus sequenceR-L-X-G-G (SEQ ID NO. 4) where X is any amino acid.

PLpro and PLPs have not only proteolytic but also deubiquitinating (DUB)and delSGylating activities that counteract the post-translationalmodification of signaling molecules that activate the innate immuneresponse. The amino acids that are responsible for these modificationsform the catalytic core of PLpro/PLP and are highly conserved. However,the overall amino acid identity in papain-like proteases is low(18-32%). The substrate binding sites in different PLpros/PLPs havedifferent preferences for various ubiquitins and Interferon StimulatedGene 15 (ISG15).

Ubiquitin (Ub) is a 76 amino acid protein that is covalently attached toa substrate via the terminal glycine residue in L-X-G-G of Ub via anisopeptide bond to a lysine or N-terminal amino group on the substrate.Substrate-conjugated ubiquitins can be further modified by additionalattachment of ubiquitins to Ub lysine residues or to the Ub N terminusto form, for example, K6, K11, K27, K29, K33, K48 and K63 linkedbranched di-ubiquitins, where K is lysine, and the number denotes theposition of the lysine within ubiquitin. Multiple conjugations are alsopossible, e.g., K48/K63, K6/K11 or combinations of K6, 11, 27, 29, 33,48, 63. Enzymes, such as PLpro, remove ubiquitin by cleaving theterminal L-X-G-G sequence that links the substrate and ubiquitin.

ISG15 is a linear bi-ubiquitin with the distinctive L-X-G-G motif at itsC terminus for attachment to target proteins. Deubiquitylating enzymes,such as PLpro, remove ISG15 by cleaving the terminal L-X-G-G sequencethat links the substrate and ISG15.

Table 1 shows the ubiquitin and ISG15 substrate preferences of somePLpros/PLPs from various viruses known to infect humans. The differencesin preferences can be used to identify the presence of a specific virus.

TABLE 1 Various human viruses and their ability to cleave ISG15 and/orvarious linear or branched ubiquitins. K48 K48/K63 K63 K6/11 ISG15Adenovirus ✓ ✓ Herpes Simplex virus 1 ✓ ✓ Human Cytomegalovirus ✓Epstein Barr Vims ✓ Kaposi Sarcoma ✓ SARS-1 ✓ ✓ ✓ SARS-2 ✓ ✓ MERS ✓ ✓ ✓NL63 ✓ ✓ Crimean Congo Hemorrhagic Fever Virus (CCHFV) ✓ ✓ ✓ ✓ DugbeVirus (DUGV) ✓ ✓ ✓ ✓ ✓ Endogenous Retrovirus Group V (ERVV) ✓

Other virus with enzymes capable of removing ISG15 and/or ubiquitin thatinfect animal hosts or plants include the infectious bronchitis virus(IBV; chicken), equine arteritis virus (EAV), porcine reproductive andrespiratory syndrome virus (PRRSV), lactate-dehydrogenase elevatingvirus (LDV; mouse), simian hemorrhagic fever virus (SHFV), Nairobi sheepdisease virus (NSDV), foot and mouth disease virus (FMDV), and turnipyellow mosaic virus (TYMV).

SARS-CoV-1, SARS-CoV-2, and MERS cause severe respiratory disease whilethe four common human coronaviruses (HCoV) 229E, OC43, NL63 and HKU1generally cause mild to moderate upper-respiratory illness in 15%-30% ofcases of common colds. For accurate diagnosis, it is essential toidentify the type of causative coronavirus. The different preferencesfor ubiquitin types and ISG15 substrates among the members of humancoronaviruses opens up the possibility of distinguishing between thestrains by screening different substrates and obtaining “fingerprints”that are distinct for each of the seven human coronaviruses, shown inTable 2.

TABLE 2 Six of seven coronavirus strains known to infect humans andtheir subgroups are listed. The cleavage of 4 different papain-likeprotease substrates is indicated by “+” where each additional “+”roughly corresponds to a 10-fold increase in activity using recombinantenzyme. L-R-G-G Ty-Dap- (SEQ ID Coronavirus Subgroup G-G NO. 1)Ubiquitin ISG15 Enzyme MERS β — + ++ +++ recombinant SARS-1 β ++ ++ +++++ recombinant SARS-2 β + + ++ +++ recombinant OC43 β 34%³ 22%² —  38%¹viral, in vivo 229E α — — — 42.4%¹ viral, in vivo NL63 α — — 24%¹ —viral, in vivo NL63 α — — +++ ++ recombinant ^(1, 2, 3)= measurementtaken after 15, 60, or 800 minutes of incubation, respectively.

The catalytic rates differ among coronaviruses and correspond to thenumber of substrates that are turned over to form product. They aregiven as Kcat (min⁻¹) in Table 3 shown before for various coronaviruses.

TABLE 3 Catalytic rates (Kcat) of various coronaviruses. Rates withwhich tetrapeptide L-R-G-G (SEQ ID NO. 1), ubiquitin and ISG15 areconverted are given in in min⁻¹. Coronavirus Tetrapeptide UbiquitinISG15 SARS-1 0.0051 10 40 SARS-2 0.3 75.9 436 MERS 0.003 18.8 32.6

Coronaviruses show a moderate mutation rate of 10⁻⁴ nucleotidesubstitutions/site/year. Twenty-seven of the proteins comprising theSARS-CoV-2 virus are mutating at different rates, with the principaltargets of COVID-19 vaccines and therapeutics, the Spike andNucleocapsid proteins, having the highest mutational variability. Incontrast, the mutation rates in SARS enzymes are low, which is expectedbecause enzymes must fold into precise three-dimensional structures tofunction. Within that structure specific amino acids are broughttogether to form substrate binding sites, catalytic sites, and bindingsites for cofactors, collectively known as active sites. These clustersof functional residues are highly conserved to ensure that only intendedsubstrates are bound and chemically modified in the intended way. Activesites of enzymes are the most highly conserved sites in proteins.Although enzymes are just as subject to random mutations as structuralproteins, mutations that change the three-dimensional structure of anenzyme or the amino acid sequence of its active sites lead to loss offunction and nonviable virus. The mutational frequency in thenonstructural proteins cleaved by PLpro—and in PLpro itself—aresignificantly lower than those observed in the spike protein (S) andnucleocapsid (N) protein.

Detection methods conventionally used for the detection of pathogensare, for example, Polymerase Chain Reaction (PCR) and antigen tests.RT-PCR is the current “gold standard” for detection of pathogens, suchas SARS-CoV-2. PCR amplifies specific DNA targets so they can bevisualized at the end of the reaction or while the reaction progressesin real-time. If the genetic material of the virus is RNA, as in thecase of coronaviruses, the RNA first has to be converted intocomplementary cDNA using reverse transcriptase (RT). The cDNA is thenused as a template for exponential PCR amplification. PCR employs twoprimers with sequences that are complementary to the target cDNA and aDNA polymerase that assembles new strands of DNA from free nucleotides.In addition to the two amplification primers, real time PCR uses a probethat binds specifically to a region of the DNA that is being amplified.The probe is labeled with a quencher and a fluorophore that fluoresceswhen the polymerase removes the probe from the DNA strand and cleavesit. The amount of fluorescence correlates with the amount of DNA beingproduced and can be observed in real time.

The relationship between SARS-CoV-2 detection, viral load, andinfectivity is not fully understood, as viral material that isdetectable by RT-PCR or antigen tests may not represent transmissiblelive virus, but prolonged shedding of non-viable viral fragments. Forexample, viable virus able to infect cell cultures was not found insamples collected after day eight of symptom onset, in spite of ongoinghigh viral loads of approx. 10⁵ RNA copies/mL that gave positive resultswith RT-PCR. In other words, the detection of viral RNA does notnecessarily mean that a person is infectious and able to transmit thevirus to another person. Information about the actual duration ofinfectivity is important so RT-PCR positive but non-infectious peoplecan return to their normal activities, correct assignments in hospitalwards can be made, and treatment options pursued. For example, lungtransplants can be made only after COVID-19 patients test negative withRT-PCR.

Current methods to determine the presence of infectious virus areindirect, time consuming, and complex. In laboratory experiments virussupernatants are transferred onto host cells, such as Vero E6 cells, andincubated for around 70 h. Cytopathogenic effects of viral infection arescored visually or by determining the remaining metabolic activity ofthe infected cells. This approach is not practical in clinical settings.

The FDA has approved over 200 SARS RT-PCR tests. RT-PCR can, in theory,detect one molecule of viral RNA, and has become the gold standard forCOVID-19 testing. Other nucleic-acid based test systems include RT-LAMP(RT loop-mediated isothermal amplification), CRISPR (Clustered RegularlyInterspaced Short Palindromic Repeats), or SPOT (Scalable and PortableTesting).

These tests share an Achilles heel: their target biomolecule is viralRNA (ribonucleic acid) that must first be transcribed intodeoxyribonucleic acid (DNA) for molecular tests to perform.Unfortunately, RNA is extremely unstable and prone to rapid degradationduring sampling, shipment, and purification. As a result, despite atheoretical analytical sensitivity of less than 5 viral copies ofpurified RNA reported for some of the RT-PCR tests, the limits ofdetection (LoD) in clinical samples vary greatly (500-80,000 copies/ml).

The COVID-19 pandemic has left approximately 176 million people infectedwith SARS-CoV-2 and over 3.8 million dead. Variants that can evadedetection are quickly emerging from the enormous reservoir of infectedpeople. New mutations in the SARS-CoV-2 genome increase RT-PCRfalse-negative test results when primer and probes bind weakly, or nolonger at all, to the mutated sequences. Although RT-PCR tests amplifycDNA from several regions of the SARS genome that generally do not allfail at the same time, one variant in France was completely undetectableby RT-PCR. But even if only one PCR product is absent, an “inconclusive”result is triggered, and the test must be repeated by a different testor sequencing.

The rate of reported false negative SARS-CoV-2 results are at between20% to 67% in clinical samples, depending on the viral load at the timeof testing. Aside from the instability of RNA, the inadequate samplecollection is another reason for high false negative rates.Nasopharyngeal (NP) swabs may miss virus, i.e., the swab may not beinserted deeply and/or broadly enough in the nasopharyngeal cavity tocapture viruses. Collection of other specimen types, such as sputum,nasal swab, and throat swab is rapid, simple, and safe but the precisionremains to be determined.

Other disadvantages of RT-PCR are:

-   -   1. RT-PCR tests require sophisticated equipment that cost        between USD 15,000-USD 90,000 and trained personnel at        centralized locations;    -   2. The test can detect inactive viral fragments remaining from a        previous infection, complicating treatment decisions that rely        on an infection-free status, e.g., for lung transplants;    -   3. Primer and probe binding are affected by changes in viral RNA        sequences, and this variability can cause mismatches between        primers and probes and the target sequence, leading to decrease        in assay performance and false negative results;    -   4. Samples contain live virus and because of the infection risk,        sampling and testing require personnel protective equipment;    -   5. It can take days for results to be reported; and    -   6. Because it contains numerous difficult-to-produce components,        such as enzymes and viral transport media, RT-PCR is difficult        to scale up.

Antigen tests are typically inexpensive and return results in minutes.Antigen tests can detect the presence of a viral protein in a biologicalsample, such as a NP swab. Generally, drops of the sample are spotted ona surface, such as a plastic well or a test strip, that is coated withone antibody that binds the SARS protein (the capture antibody) andanother that detects it (the detection antibody). If both antibodiesbind, a signal is generated, such as a color change, that can be readvisually although some setups use small readers to improve the accuracy.The challenge with antigen tests is finding antibodies that both bind tothe single viral protein, such as the SARS-CoV-2 spike protein, atseparate sites, but do not cross react with other coronaviruses or bindnonspecifically.

Weak signals are a major drawback because antigen tests do not amplifythe protein signal; therefore, they are inherently less sensitive thanRT-PCR or enzyme assays. As a result, most antigen tests have adetection sensitivity of between 50% and 90%, relative to RT-PCR.

In addition to a high false-negative rate of detection, antigen testsshare some of the disadvantages of RT-PCR tests, namely that they candetect inactive virus and the capture and/or detection antibody may nolonger bind when viral antigens mutate.

Serological tests detect antibodies developed against the SARS-CoV-2 inthe blood of infected people. These tests can help identify individualswho have mounted an immune response to the virus as part of either anactive or prior infection. Antibodies against SARS-CoV-2 take 1-3 weeksto develop in the bodies of those infected; therefore, detecting anantibody response is not suitable for diagnosing active infections. Inaddition, the false-positive detection rate through antibody binding tosimilar proteins from other coronaviruses is possible and remains to bedetermined. Further, there is not enough information yet to determinewhether antibody-positive individuals will be immune and protected fromreinfection and therefore be safe to reenter society without spreadingthe disease. Moreover, recent legal rulings prohibit employers fromrequiring employees to take serological tests as a condition forreturning to work following possible exposure because the tests cannotunambiguously identify healthy from ill people. What is needed is amethod for detecting pathogens that on-site, rapid, reproducible withlarge volumes of samples, and is accurate.

Natural, therapeutic, or vaccine-derived antibodies can block(“neutralize”) pathogens from infecting host cells by binding toproteins on the outside of pathogens that interact with host cellreceptors. For example, antibodies against the SARS spike proteinprevent the virus from binding to ACE2 receptors and from entering acell. The antibody recognizes a specific amino acid sequence on thetarget protein (the antigen). Therefore, mutations in pathogen antigenscan reduce or abolish antibody neutralizing activities when antibodiesbind more weakly or no longer at all to the antigens.

Although COVID-19 vaccines have shown up to 95% efficacy in preventinginfection with wildtype SARS-CoV-2, reports show that the South Africanvariant has complete immune-escape in South African convalescent serumsamples, and reductions in neutralizing activity in vaccinee serumsamples for all four vaccines tested have been reported. Even moredisturbing is the situation in Manaus, Brazil, where a second wave ofinfection due to a variant swept through a population that was already76% seropositive owing to prior infection in the spring of 2020. Escapevariants appear to be responsible for the surge in infections in someIndian cities where around 50% of the population had already beeninfected. Eight mutations in the spike protein have already beenidentified that are suspected of reducing the neutralizing activity ofhuman antibodies raised against it by vaccines or natural immunity.

Therefore, it is expected that annual booster shots will be needed forCOVID-19, like for the flu. Although large-scale Phase 3 clinical trials(including approx. 30,000 subjects) to test the safety and tolerabilityare not required for booster vaccines, the FDA still requires smallclinical trials with around 300 subjects to ensure that the new vaccineshave neutralizing activities. Vaccine developers test the efficacy ofnew vaccines against seasonal circulating strains by first measuringtheir neutralizing activity in the lab, and later in clinical trials. Inthe lab, virus—antibody mixtures are transferred onto host cells,incubated for around 70 h and cytopathogenic effects of viral infectiondetermined. In clinical trials, the efficacy of the vaccine isdetermined by comparing the number of cases between the groups thatreceived placebo or vaccine. Therefore, what is needed is a method forquickly detecting pathogens to more rapidly test vaccine efficacy.

Drugs that can inhibit the activity of pathogen enzymes and therebyprevent viral replication are promising therapeutics. The majority ofhigh throughput or high content drug screens are performed using definedreaction conditions and recombinant enzymes in biochemical assays. Thedrawbacks of biochemical assays are that drugs with cytotoxic effectsand/or poor cell permeability can be missed. Potential drawbacks ofcell-based expression systems are that recombinant enzymes often behavedifferently than natural viral enzymes and exhibit different inhibitionprofiles. The spread of SARS-CoV-2 has stimulated the development ofsmall molecule antiviral inhibitors to treat patients who do not acceptor respond to vaccines. Drugs that can inhibit the activity of SARSenzymes and thereby prevent viral replication are promisingtherapeutics.

An affordable, simple, rapid, and on-site test such as the testaccording to the present invention is needed to identify and containinfections immediately, and thereby prevent further infections andmitigate an outbreak. What is also needed is a method to acceleratecell-based drug discovery by measuring the potencies of inhibitorsagainst natural, unmodified enzymes. What is further needed is a methodto screen scanning libraries using crude active lysates from culturedcells to quickly and efficiently identify peptides cleaved by an enzymeof interest with the least cleavage from endogenous host enzymes.

BRIEF SUMMARY OF EMBODIMENTS OF THE PRESENT INVENTION

The present invention relates to a method for identifying a pathogen,the method comprising: collecting a specimen, wherein the specimencomprises a cell; lysing the cell to form a cell lysate, wherein thecell lysate comprises an internal control; contacting the cell lysatewith a substrate, wherein the substrate comprises a sequence of anatural pathogen protein that may be cleaved by a pathogen enzyme andwherein the substrate comprises a signaling moiety; cleaving thesubstrate; and simultaneously reading a signal from the substrate andinternal control. In one embodiment, the specimen is a tongue scrape.

In another embodiment, the pathogen is a coronavirus. In anotherembodiment, the coronavirus is SARS-CoV-2. In another embodiment, thesubstrate is a peptide. In another embodiment, the peptide is 4 aminoacids in length. In another embodiment, the peptide comprises thesequence L-R-G-G (SEQ ID NO. 1).

In another embodiment, the internal control comprises a peptideconjugated to a signaling moiety that produces a signal that can bedifferentiated from the signal of the substrate. In another embodiment,the internal control comprises ACE2.

In another embodiment, the pathogen enzyme is a protease. In anotherembodiment, the protease is PLpro.

In another embodiment, the method further comprises a calibrator. Inanother embodiment, the calibrator calibrates fluorescent andunconjugated signaling moieties. In another embodiment, the calibratorquantifies the amount of pathogen present in the cell lysate.

In another embodiment, the cleavage substrate is read in about 1 min toabout 15 min. In another embodiment, the method is performed as alateral flow assay. In another embodiment, the method further comprisescleaving the substrate to produce one peptidic and one non-peptidicfragment. In another embodiment, the method further comprisescalculating the rate of substrate cleavage to identify the pathogen.

In another embodiment, the substrate comprises ubiquitins. In anotherembodiment, the cell lysate is crude. In another embodiment, thesubstrate comprises biological fragments and a 4-amino-acid peptidesequence.

In another embodiment, the method further comprises identifying acoronavirus variant from the substrate cleavage. In another embodiment,the signal indicates the presence of an active viral infection. Inanother embodiment, the signal indicates the presence of antibodies.

In another embodiment, the substrate comprises ubiquitin, ISG15, and apeptide comprising the amino acid sequence L-R-G-G (SEQ ID NO. 1). Inanother embodiment, the substrate is comprised within a competitiveassay. In another embodiment, the ubiquitin and the ISG 15 are contactedwith the peptide comprising the amino acid sequence L-R-G-G (SEQ ID NO.1). In another embodiment, the ubiquitin and the ISG15 are conjugated toa substrate that comprises the amino acid sequence L-R-G-G (SEQ ID NO.1).

In another embodiment, the substrate is derived from a mammal. Inanother embodiment, lysing the cells comprises contacting the cells withlysis buffer.

In another embodiment, the method of the present invention is easy touse, does not require specialized personnel or equipment, and has aminimal burden on logistics. The method may comprise reagents that maybe chemically synthesized within days in bulk for millions of tests,allowing for widespread screening that helps prevent pathogentransmission and mitigates pathogen outbreaks.

In another embodiment, the method provides a simple positive or negativeresponse and/or reading in less than 15 minutes. The method may be usedto detect pathogens in humans, animals, and/or plants.

In another embodiment, the method comprises a signal generated when apeptide substrate is cleaved by a protease to produce one peptidic andone nonpeptidic fragment. The nonpeptidic fragment corresponds to thefluorophore that is now fluorescent.

In another embodiment, the method comprises an enzymatic assay. Theenzymatic assay may detect cleaved substrate by fluorescent signals. Theenzymatic assay may comprise a substrate labeled with a reagent and maybe added directly to live cells or crude cell lysate. The substrate mayenter cells or contact crude cell lysate and may be cleaved byendogenous enzymes. Cleavage may be observed in real time by monitoringthe fluorescence increase. The substrate may be four amino acids inlength and the enzyme may be PLpro. The enzymatic assay may furthercomprise an internal control. The internal control may comprise ACE2. Inanother embodiment, the substrate may be derived from different speciesto identify the pathogen.

Objects, advantages and novel features, and further scope ofapplicability of the present invention will be set forth in part in thedetailed description to follow, taken in conjunction with theaccompanying drawings, and in part will become apparent to those skilledin the art upon examination of the following, or may be learned bypractice of the invention. The objects and advantages of the inventionmay be realized and attained by means of the instrumentalities andcombinations particularly pointed out in the appended claims.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The accompanying drawings, which are incorporated into and form a partof the specification, illustrate one or more embodiments of the presentinvention and, together with the description, serve to explain theprinciples of the invention. The drawings are only for the purpose ofillustrating one or more embodiments of the invention and are not to beconstrued as limiting the invention. In the drawings:

FIG. 1 is a schematic showing a quench mechanism based on electrontransfer between donor and acceptor peptide SEQ ID NO. 1;

FIG. 2 is a graph showing a fluorescence increase through cleavage ofthe electron transfer substrate in crude tongue scrape lysate to whichrecombinant PLpro was added;

FIG. 3 is a schematic showing of a quench mechanism based on FoersterResonance Energy Transfer (FRET), where a peptide composed of sevenamino acids (circles) (SEQ ID NO. 2) is labeled on one end with thefluorophore fluorescein isocyanate and on the other with a quencherdinitrophenyl (DNP);

FIG. 4 is a graph showing fluorescence increase through cleavage of theFRET substrate;

FIG. 5 is a graph showing the simultaneous measurement of peptides uponcleavage by endogenous ACE2 and recombinant PLpro;

FIG. 6 is a graph showing fluorescence of the FRET substrate of FIG. 3in fresh crude lysate or in crude lysate that was stored at 4° C. for 2hours;

FIG. 7 is a diagram showing the test procedure to detect a pathogen;

FIG. 8 is a graph showing serial dilutions of unconjugated7-amino-methylcoumarin (AMC);

FIG. 9 is a schematic of a lateral flow assay based on the SARS proteasecleavage assay;

FIG. 10 is a graph showing cleavage of peptides with the amino acidsequence L-R-G-G-AMC (SEQ ID NO. 5) or Tyr-Dap-G-G-AMC in the presence(+) or absence (−) of crude tongue scrape lysate;

FIG. 11 is a graph showing an enzyme concentration curve usingrecombinant SARS PLpro and ISG15-AMC (circles) or L-R-G-G-AMC (SEQ IDNO. 5) (triangles);

FIG. 12 is a graph showing an enzyme concentration curve usingrecombinant NL62 PLP2 and Ubiquitin-AMC (circles), ISG15-AMC (squares),or tetrapeptide-AMC (triangles);

FIG. 13 is a graph comparing ISG15-AMC cleavage between crude lysatesfrom infected human cells and uninfected lysates;

FIG. 14 is a schematic showing a peptide combination that results insuperior sensitivity conferred by the ISG15 substrates and specificityconferred by the tetrapeptide sequence Tyr-Dap-G-G;

FIG. 15 is a graph showing the cleavage of substrates Tyr-Dap-G-G,L-R-G-G (SEQ ID NO. 1), ubiquitin (UbiQ) and ISG15 in crude lysates frominfected human cells;

FIG. 16 is a graph showing cleavage of the ACE2-FITC substrate in crudelysates from infected human cells; and

FIG. 17 is a graph showing florescence from the substrate L-R-G-G-AMC(SEQ ID NO. 5) and various concentrations of recombinant SARS PLproenzyme.

DETAILED DESCRIPTION OF THE INVENTION

An embodiment of the present invention is directed to a method for anactivity-based diagnostic (ABDx) for a rapid response platform capableof detecting pathogens by measuring the activities of enzymes that areunique for a given pathogen, for example, papain-like protease (PLpro)in SARS coronavirus-infected cells.

The term “enzyme” means any enzyme known to those skilled in the art. Anenzyme may comprise a viral, prokaryotic, or eukaryotic enzyme. Anenzyme may be present in a solution and/or biological solution,including, but not limited to, a tongue scrape cell mixture, saliva,sputum, blood, lymph, urine, feces, bodily fluid, or a combinationthereof. The enzyme may be present in an extract. The extract maycomprise, but is not limited to, any pathogen-infected cell, an extractof plants, bacterial or fungal cultures, or any biological extract.

The term “protease” means proteins that are known as proteinases orpeptidases. Proteases are classified and/or defined on the basis oftheir catalytic mechanism into the following groups: serine proteases(S), cysteine proteases (C), aspartic proteases (A), metalloproteases(M), and Unknown, or as yet unclassified, proteases (U). Papain-likeproteases (PLpro) belong to the class of cysteine proteases.

The term “substrate” means both naturally occurring substrates, andsynthetic analogues of naturally occurring substrates comprising asubstrate recognition sequence. Enzyme substrates mean any moleculecomprising proteins, peptides, deoxyribonucleic acid (DNA), ribonucleicacid (RNA), lipids, carbohydrates, polyesters, polythioesters,polyisoprenoids, or a combination thereof. A substrate may be derivedfrom a biological source. The biological source may comprise a mammal, areptile, a bird, or a combination thereof. The mammal may comprise anymammalian species including, but not limited to, a bat, a sheep, ahuman, a pig, a cow, a goat, or a combination thereof.

The term “peptide” and “protein” may be used interchangeably and maycomprise amino acid sequences of any length. The terms “peptide” and“protein” comprise molecules with naturally occurring, semi-synthetic,or artificial sequences, where amino acids may be comprised withinartificial sequences that do not naturally occur in proteins. Forexample, the terms “peptide” and “protein” may refer to an amino acidsequence of a recombinant or non-recombinant peptide with an amino acidsequence of a native peptide; a biologically active fragment of a nativepeptide; a biologically active peptide analogue of a native peptide; abiologically active variant of a native peptide; a peptide having anartificial sequence comprising a biologically active consensus sequence;a peptide having a wholly artificial sequence; or a combination thereof.In general, the terms “protein”, “peptide” and “amino acid” are to beinterpreted broadly to encompass any derivative molecules that provideone or more bonds capable of being hydrolyzed by proteolytic enzymes.These hydrolysable bonds may be provided within the structure of themolecule itself or alternatively or additionally may be formed when themolecule is labelled by the attachment of a marker.

The term “pathogen” means any infectious agent that can produce disease.Pathogens may comprise, but are limited to, viruses, bacteria,protozoans, prions, viroids, or fungi. Viruses may include, but are notlimited to, coronaviruses. Coronaviruses may include, but are notlimited to, SARS-CoV-1, SARS-CoV-2, and MERS.

The term “host cells” means any cell that a pathogen can invade directlyor indirectly. Direct entry often involves attachment of a pathogen tospecific receptors on a host cell and indirect entry may occur throughfusion with the host cell membrane.

The term “quencher” means any chemical or biological substance known tothose skilled in the art that is capable of absorbing energy, and thusquenching fluorescence from a fluorophore. A quencher may comprise amidebonds, which withdraw electrons from some fluorophores. Fluorophores maycomprise, but are not limited to, aminomethyl coumarin (AMC),aminocarbomylcoumarin (ACC), Rhodamine 110, or a combination thereof.Withdrawal of electrons from fluorophores may result in fluorescencequenching in the intact substrate.

The term “crude” means a solution, sample, specimen, cell lysate, orcombination thereof that is produced immediately after contact withlysis buffer. A crude solution, sample, specimen, cell lysate, orcombination thereof are not subjected to processing steps other thancontact with lysis buffer. Processing steps include, but are not limitedto, centrifugation, sonification, extraction, separation, or acombination thereof.

The method may provide a simple positive or negative response and/orreading in less than 15 minutes. A positive or negative response and/orreading may be provided in at least about 1 min, about 1 min to about 3min, about 3 min, to about 5 min, about 5 min to about 7 min, about 7min to about 9 min, about 9 min to about 11 min, about 11 min to about13 min, about 13 min to about 15 min, or about 15 min. The method mayprovide a platform for the detection of various existing and emergingpathogen-mediated disease in humans, animals, and plants.

The method may be used to measure pathogen activity. The signalgenerated by the method may indicate the presence of natural,therapeutic or vaccine derived antibodies. The presence of antibodiesmay neutralize the viral activity and reduce the signal. The presence ofpathogen activity may also indicate an active infection. The level ofpathogen activity may indicate the severity of infection, with greaterpathogen activity indicating a greater degree of infection.

The method may comprise a signal generated when a peptide substrate iscleaved by a protease. The signal may be a fluorescence signal that isquenched in an intact substrate by the electron withdrawing propertiesof an amide bond that connects a C-terminal amino acid with thefluorophore. When the peptide bond between the terminal amino acid andthe fluor is cleaved, one peptidic and one nonpeptidic fragment may beproduced. The nonpeptidic fragment corresponds to the fluorophore thatis now fluorescent. The principle of the electron transfer quenchingassay of the present invention for SARS testing is shown in FIG. 1 .

The enzyme in the enzyme activity assay, i.e., the protease, may not beconsumed in the cleaving process. The enzyme may be available tocontinue cleaving sites until all enzyme-substrate binding sites havebeen saturated, effectively amplifying the signal. This process is shownin FIG. 2 by the steady increase of fluorescence over time.

The fluorescent signal may be quenched by a mechanism termed FoersterResonant Energy Transfer (FRET). In FRET, photons emitted by afluorophore upon excitation by a light source (e.g., mercury lamp,laser, or LED) are absorbed by a quencher that “turns off” fluorescence.Energy transfer from the fluorophore to the quencher may be highlydistance dependent and occurs only up to a distance of approximately 10nm (corresponding to approximately 28 amino acids). Therefore, when aFRET peptide is cleaved by a protease, fluorescence is “turned on” asthe quencher and fluorophore move apart. The principle of a FRET assayof the present invention that may be used as an internal control forSARS testing is shown in FIG. 3 . The FRET peptide may be recognized byhuman angiotensin converting enzyme 2 (ACE2) that is ubiquitouslyexpressed on epithelial cells in the oral cavity through which SARScoronaviruses and other viruses enter cells. The control confirms thatthe sample collected has sufficient quantity and quality for the methodto perform properly. As an example of the function of the internalcontrol, a fluorescence increase of ACE2 substrate as a function of thelysate concentration is shown in FIG. 4 .

The method may comprise using a substrate. Substrates are generallyspecific for one enzyme. Substrates may comprise but are not limited to,DNA, RNA, protein, peptides, or a combination thereof. The peptide maycorrespond to the amino acid sequence of a pathogen protein. Substratesmay comprise a signaling moiety. The signaling moiety may be attached tothe substrate. Moiety attachment may be by conjugation. The signalingmoiety may produce a fluorescent, colorimetric, or chemiluminescentsignal. The substrate may comprise a gene fragment, ubiquitin, aminoacids, or a combination thereof. The substrate may comprise one or morebiological fragments. The biological fragments may comprise genefragments, ubiquitin fragments, or a combination thereof. The gene maycomprise ISG15. The substrate may comprise ubiquitin. Ubiquitin may beattached to the substrate. The ubiquitin may be attached by conjugation.The ubiquitin may be naturally attached to the substrate. Substrateswith unnatural amino acids may be used to confer specificity for anenzyme and may be added to fragments of ubiquitin and ISG15 separatelyor as a conjugate.

The substrate may be cleaved by a protease. The protease may be apathogen protease. For example, substrates that are cleaved bycoronavirus papain-like proteases are based on the amino acid sequencemotif R-L-X-G-G (SEQ ID NO. 4). The substrate may comprise the aminoacid sequence R-L-X-G-G (SEQ ID NO. 4), L-R-G-G (SEQ ID NO. 1), or acombination thereof. Ubiquitin and ISG15 with a terminal L-R-G-G (SEQ IDNO. 1) sequence may be added in a competitive assay where the substrateis a labeled tetrapeptide based on the L-R-G-G (SEQ ID NO. 1) sequence.Ubiquitin and ISG15 without the C-terminal L-R-G-G (SEQ ID NO. 1)sequence may be generated and added separately to a tetrapeptide basedon the L-R-G-G (SEQ ID NO. 1) sequence. Ubiquitin and ISG15 substrateswithout the C-terminal L-R-G-G (SEQ ID NO. 1) sequence may be added inseveral fragments and added separately to a tetrapeptide based on theL-R-G-G (SEQ ID NO. 1) sequence.

Substrates may be derived from different species to identify a pathogen.Substrates may be preferentially cleaved by a pathogen depending on thespecies the substrate sequence is derived from. For example, SARS-CoV-1does not cleave ISG15 from fish or camel, whereas SARS-CoV-2 cleavesISG15 from fish and MERS cleaves ISG15 from camel. Thus, substrates fromdifferent species may identify specific pathogens including, but notlimited to, coronaviruses.

Screening combinatorial substrate libraries containing a wide variety ofnonproteinogenic amino acids allows to further define substrates thatare recognized only by one protease and not by another within a family.For example, peptides may be specifically cleaved by SARS PLpro but notby MERS PLpro or human deubiquitylating enzymes. Peptides specificallycleaved by SARS PLpro have the following sequences: Ac-Abu(Bth)-Dap-G-G,Ac-hTyr-Phe(guan)-G-G, Ac-Tyr-Dap-G-G, and Ac-hPhe-Dap-G-G. Ubiquitinand Interferon Stimulated Gene15 are other substrates that arerecognized by papain-like proteases and cleaved at their carboxyterminalL-X-G-G amino acid sequence.

Modifications to substrates affected by enzymes may comprise, but arenot limited to, phosphorylation, dephosphorylation, methylation,demethylation, ubiquitinylation, deubiquitylation, glycosylation,acetylation, hydroxylation, addition, and removal of InterferonStimulating Gene 15 (ISG15), or a combination thereof. Enzymes may alsoaffect cleavage of their substrates.

The method may detect any pathogen variant. False-negative test resultsfrom “variants” with mutations in enzyme active sites are exceedinglyunlikely to occur because enzyme active site mutations would reduce orabolish pathogen function and are not compatible with survival and/ortransmission. In contrast, variants with mutations in other regions ofthe viral genome that do not interfere with enzyme active sites will notaffect the ability of a test to detect pathogens.

The method may directly measure the efficacy of natural, therapeutic, orvaccine-derived antibodies by detecting the activity of enzymes frompathogens that were able to enter a host cell. Therefore, the method ofthe present invention may be used as a portable companion diagnosticstest. Subjects enrolled in pharmaceutical clinical trials that test theefficacy of therapeutic or vaccines-derived antibodies may be given acomplete test system and report daily test results directly to theclinical trial center, for example, by a mobile phone and/or mobilephone application.

The method may accelerate cell-based drug discovery by measuring thepotencies of inhibitors against natural, unmodified enzyme. The presentinvention may be applied to screen peptide libraries using crude activelysates from cultured cells to quickly and efficiently identify peptidesthat are cleaved by the enzyme of interest with the least cleavage fromendogenous host enzymes.

The method may comprise using an enzymatic assay. The enzymatic assaymay comprise a fluorescence-based assay system using a substrate labeledwith a quencher and a fluorophore which together form a Foerster (orFluorescence) Resonance Energy Transfer (FRET) pair. The emission may bequenched by the quencher in an intact substrate and liberated uponcleavage. Fluorophores, may comprise, but are not limited to,amino-methylcoumarin (AMC), amino carbomylcoumarin (ACC), Rhodamine 110,or a combination thereof. Fluorophores may be quenched by the electronwithdrawing properties of the amide bond between the terminal amino acidand the fluorophore of the substrate. Fluorophores may not require aseparate quencher to be quenched.

Formats for performing enzymatic assays may comprise, but are notlimited to, wells of multi-well plates, vials, cuvettes, microscopeslides, test strips, or a combination thereof. The fluorescenceliberated after cleavage may be detected in any device capable ofproviding excitation light, e.g., an LED, mercury lamp, or laser, andrecording emission. These devices may comprise, but are not limited to,battery-operated handheld fluorometers containing LEDs and appropriatephotodiodes, benchtop plate-based fluorometers, fluorescence activatedcell sorters (FACS), fluorescence microscopes, real time polymerasechain reaction (RT-PCR) machines, quantitative real time polymerasechain reaction (Q-PCR), or a combination thereof.

The enzymatic assay may comprise a membrane permeable enzyme reportersubstrate. The enzyme reporter substrate may be labeled with a reagent,including but not limited to, Rhodamine 110. The enzyme reportersubstrate may be added directly to live cells. The enzyme reportersubstrate may enter cells and may be cleaved by endogenous enzymes.Cleavage may be observed in real time by monitoring the fluorescenceincrease in a fluorescence microscope, fluorometer, or any other devicecapable of detecting fluorescence.

Cleavage of substrates may also be detected using lateral flow assays(LFA) where portions of substrates are captured on the matrix byimmobilized capture reagents. The Capture reagent may comprise, but isnot limited to, streptavidin, antibodies, or a combination thereof.Captured substrate portions, i.e., captured analytes, may be detectedusing detection antibodies with conjugated chromogens, metal particles,or a combination thereof.

The enzymatic assay may comprise a chromatography technique. Thechromatography technique may comprise, but is not limited to, sizeexclusion chromatography, gel-based separation, fluorescencepolarization, or a combination thereof to detect the change in the sizeof the reporter substrate. The enzymatic may also comprise substrateslabeled with an electrochemically active label that records cleavageactivity.

The enzymatic assays may be homogeneous or heterogeneous. Homogeneousenzymatic assays may be of the add-and-mix type. Heterogeneous enzymaticassays may comprise steps including, but not limited to, precipitationand/or separation. In homogeneous enzymatic assays, the progress of areaction may be monitored in real time as the reaction occurs or at thebeginning and end of the reaction. Enzymatic assays may measure theactivity of enzymes of a pathogen or an infectious agent capable ofinducing a selective alteration in enzyme activity in the host.

The method may comprise a fluorescent dye. The fluorescent dye may actas a fluorophore. The excitation and emission properties of thefluorophore may fall within any measurable wavelength range, forexample, an absorption range between 300 nm to 800 nm and emissionrange, for example, between 350 nm to 800 nm. The fluorescent dye mayfall into various classes, where combinations of fluorescent dyes may beused in the same class or between different classes. The classes of thefluorescent dye may comprise, but are not limited to, xanthene dyes,e.g., fluoresceins and rhodamines; coumarins, e.g., umbelliferone;benzimide dyes, e.g., Hoechst 33258, phenanthridine dyes e.g., Texas Redand ethidium dyes; acridine dyes; Bodipy; cyanine dyes, e.g., thiazoleorange, thiazole blue, Cy 5, and Cyfr; carbazole dyes; phenoxazine dyes;porphyrin dyes; quinoline dyes; or a combination thereof. Thefluorescent dye may absorb light in the ultraviolet, visible, orinfrared wavelength ranges or a combination thereof.

The method may comprise an organic dye. Organic dyes may be used asquenchers in FRET assays if the organic dye is spectrally matched withthe fluorophore. The method may also comprise a quencher. Quenchers maybe fluorescent themselves or be dark quenchers with little or nointrinsic fluorescence. Dark quenchers may comprise, but are not limitedto, 4-(4′dimethylaminophenylazo)benzoic acid (DABCYL). Quenchers maycomprise black hole quenchers. Black hole quencher may comprise diazodyes of the BHQ series. Black hole quenchers may provide a broad rangeof absorption which overlaps well with the emission of manyfluorophores. Quenchers may be directly bonded to the substrate.Quenchers may be bonded indirectly to substrate, for example, bymetal-ion association to a phosphate group present on the substrate.Metal ions capable of bonding to phosphate-labeled substrates andquenching the fluorescence of a reporter fluorophore may comprise, butare not limited to, divalent and/or trivalent metal ions. The divalentand trivalent metal ions may comprise, but are not limited to, iron,gallium, zirconyl chloride, manganese chloride, or a combinationthereof.

Peptides cleaved by PLpro and ACE2 may be labeled with differentfluorophores that have non-overlapping excitation and emissionproperties. This allows both peptides to be measured simultaneously inone sample, as shown in FIG. 5 .

The method may comprise a specimen. The specimen may comprise, but isnot limited to, a tongue scrape, saliva, sputum, blood, urine, nasal andanal swabs, mucus, serum, plasma, urine, spinal fluid, tissue biopsy,vaginal fluid, amniocentesis fluid, tears, bronchoalveolar fluid, otherfluid or tissue or cells, or a combination thereof. The specimen maycomprise any cell type. The specimen may also comprise crude lysatesfrom cells that are grown in culture. Crude lysates from cells that aregrown in culture were used in the experiment results shown in FIG. 13 ,FIG. 15 , and FIG. 16 .

Tongue scrape specimen samples may comprise an enrichment of cellular,potentially virus-infected material in cells from the tongue. Hundredsof viruses may be present in each infected cell, and around 30 millioncells/cm² are collected in a tongue scrape. Lysate derived from aspecimen may be used directly without any further processing, e.g.,filtration, or centrifugation. Crude cell lysate may be stable for atleast about 2 hours, about 2 hours to about 4 hours, about 4 hours toabout 6 hours, about 6 hours to about 8 hours, about 8 hours to about 10hours, about 10 hours to about 12 hours, or about 12 hours on ice. Celllysate may be stable for at least about 2 days, about 2 days, to about 5days, about 5 days to about 10 days, about 10 days to about 15 days,about 15 days to about 20 days, about 20 days to about 25 days, about 25days to about 30 days, or about 30 days when frozen at about −20° C. toabout −80° C.

The method may comprise using a kit for detecting protease activity incrude lysates from cells. The kit may comprise a specimen collector. Thespecimen collector may comprise a tongue scraper, a transfer pipette, acollection vial comprising lysis buffer, a vial comprising assaycomponents in liquid or freeze-dried form, or a combination thereof.Optionally, the assay components may be in a form to remain activewithout specialized storage conditions.

The method may comprise a multiplex test to detect PLpro and ACE2simultaneously. The multiplex test may be used in combination with afluorometer. The fluorometer may be a commercial fluorometer and may bebattery-operated and/or handheld. The multiplex test may comprisecollecting a specimen and at least partially disposing the specimen in aspecimen vial. The specimen may be a tongue scrape. The specimen vialmay comprise a lysis buffer that disrupts the lipid membrane of cellsand viruses. A solution may be formed by contacting the specimen withthe lysis buffer. The solution may be transferred to a vial thatcontains all components for the test in a stable form. The vial may beat least partially disposed within a fluorometer. The fluorometer may beconfigured to measure emission from the positive control ACE2 peptideand the second to measure emission from the PLpro peptide. The channelsmay be switched by the push of a button.

The method may comprise test reagents and sampling materials sufficientfor a single measurement. Alternatively, the method may comprise enoughreagents for hundreds to thousands of measurements. Measurements may beperformed in multi-well plates, including but not limited to, 96-well,384-well, and/or 1536-well plates for high content screening (HCS)and/or high throughput screening (HTS) applications.

Optionally, the method may comprise a calibrator. The calibrator mayallow for semi-quantitative determination of the viral load. Thecalibrator may comprise serial dilutions of unconjugated and unquenchedfluorophore. As an example, FIG. 8 shows a linear calibrator responsethat is 50% broader than the expected response from samples.

The quantity of specimen may be normalized to the measured signalmeasured allowing for the quantification of the pathogen load. Forexample, total protein can be rapidly measured in solution by theinteraction of a fluorophore with the amines of polypeptides. Threereadings of each sample may be taken, including the fluorescence derivedfrom PLpro peptide cleavage; the fluorescence derived from ACE2 peptidecleavage; and the fluorescence of an amine-binding protein quantifyingdye added to the sample after the enzymatic reactions are completed.Other reagents can be used to quantify the specimen lysate. These otherreagents may comprise, but are not limited to, reagents that measureDNA, e.g., with Hoechst Dye 33258, RNA, any other common biological incrude lysate, or a combination thereof.

The fluorescence from samples may be taken immediately and thereafter atregular or semi-regular intervals. Typical interval periods may be atleast about 30 sec, about 30 sec to about 1 min, about 1 min to about2.5 min, about 2.5 min to about 5 min, about 5 min to about 10 min,about 10 min to about 15 min, or about 15 min. The difference inrelative fluorescence units (RFU) between the time 0 reading and thefinal endpoint may be calculated (delta RFU).

A large delta RFU from the PLpro peptide may indicate the presence of apathogen and a large delta RFU from the ACE2 peptide, the internalpositive control, may indicate successful sampling, lysis, and assayperformance. Delta RFU from the ACE2 peptide only indicates the absenceof pathogen infection, and absence of delta RFU from the ACE2 peptideindicates an inconclusive test.

The method may be an activity-based diagnostics approach intended forsemi-quantitative and/or quantitative detection of SARS PLpro activityin crude lysates collected from cells in the oral cavity. The method maybe used to test individuals suspected of having COVID-19 or for use inindividuals without symptoms or other epidemiological reasons to suspectCOVID-19, using supervised or unsupervised sample collection. Testingmay be performed at point of care (POC) sites or in patient caresettings operating under a Clinical Laboratory Improvement Amendments of1988 (CLIA) certificate or at sites that can perform low complexitytests approved for waiver under the Clinical Laboratory ImprovementAmendments of 1988 (CLIA). CLIA-certified sites may includelaboratories, hospitals, or any other centralized testing site.CLIA-waived sites may include pharmacies, doctor's offices, bordercontrol agencies, airports, schools, colleges, businesses, or personalresidences.

The method may comprise using any device capable of fluorescenceexcitation and emission measurement suitable as the measurement deviceof the current invention. The device may comprise, but is not limitedto, a battery-operated instrument, a handheld instrument, a benchtopplate-based fluorometer, a fluorescence activated cell sorter (FACS), afluorescence microscope, an RT-PCR machine, a Q-PCR machine, orcombination thereof. Testing using fluorescence microscopes may comprisedeposition of specimens into well-based microscope slides. Testing usingRT-PCR machines may comprise programming the instrument to cycle at onetemperature, e.g., at about room temperature.

The method may comprise a lateral flow assay (LFA). Activity-baseddiagnostics approach may be used for detection of cleavage using LFAs.In this application, biotinylated substrates and fragments may becaptured on the matrix by immobilized capture reagents. The reagents maycomprise streptavidin. The cleaved fragment may be detected usingdetection antibodies that are labeled with chromogens or metalparticles. An example of cleaved fragment detection using avidin andantibodies is shown in FIG. 9 .

The method may comprise using a viral enzyme as a biomarker of apathogen. Viral enzymes are essential for survival and “variants” thatlead to loss of function are not viable. These viral enzymes maynaturally be present in cell lysate. Therefore, enzyme-activity basedtests have reduced false-negative results due to variants compared toother methods. Enzyme-activity based tests provide a generic platformfor detecting known and new variants that may evade detection by othermethods such as RT-PCR and escape immunity from neutralizing antibodies.

The method may be specific to a certain substrate. For example, MERSPLpro and human UCH-L3 do not recognize the substrate hTyr-Dap-G-G usedin the present invention to measure SARS PLpro activity and this wasconfirmed using crude tongue scrape lysate as shown by FIG. 10 ; orusing crude lysates from cells infected with coronavirus 229E, as shownin FIG. 15 . Although tetrapeptides may be converted in crude lysatesfrom cells infected with 0043 coronavirus, the cleavage was observableonly after 60 or 800 minutes.

ISG15 and/or ubiquitin binding in the SARS PLpro substrate binding sitemay be distant from the active site where the L-R-G-G-based peptide (SEQID NO. 1) is bound. The method may combine the sensitivity-conferringISG15 and/or ubiquitin protein (without the tetrapeptide cleavagesequence) and the specificity-conferring readout peptide Tyr-Dap-G-G-AMC(where AMC=aminomethyl coumarin) to obtain a substrate mixture thatconfers superior sensitivity and specificity to SARS PLpro. For example,the ISG15 and/or ubiquitin peptide without the terminal L-R-G-G (SEQ IDNO. 1) sequence may be combined with the substrate Tyr-Dap-G-G-AMC toSARS coronavirus PLpro before, later, or at the same time. More than twofragments of a specific substrate may also be added. Optionally, onesubstrate may be produced that comprises the ISG15 and/or ubiquitinsequence and the specificity-conferring sequence.

The method may be used to determine optimal cleavage using ISG15 andubiquitin substrates from different species. Monitoring the slopes ofreaction progress over time gives an estimate of the catalytic rates,which may provide a further differentiator between the pathogens, i.e.,viruses, bacteria, and/or coronaviruses, to identify them. For example,PLpro from SARS-CoV-1, SARS-CoV-2 and MERS all cleave tetrapeptide,ubiquitin, and ISG15 but at substantially different rates. Thedifferences in catalytic conversion of ubiquitin and ISG15 betweenSARS-CoV-1 and SARS-CoV-2 are expected to allow distinguishing betweenthe SARS strains.

Quantification of ACE2 measurement may be predictive of the risk forbecoming infected with SARS viruses as these coronaviruses enter cellsthrough ACE2. A high expression may be indicative of a higher risk forinfection than a low expression. Quantification is achieved bydetermining the protein amount present in the sample and calculating theamount of protein relative to the activity of ACE2. In addition,upregulation of ACE2 might be an independent marker of SARS infection.The enzymatic assay results in FIG. 16 show that ACE2 is an independentmarker of SARS infection in in vivo studies using human tissue culturedcells infected with coronavirus 229E.

The method may comprise using a rapid cell lysis protocol with whichcells from human tongue scrape are immediately lysed. The rapid celllysis protocol may comprise using lysis buffer. The lysis buffer maykeep enzymes in the active form and enzyme activity may be measured forat least about 2 hours, about 2 hours to about 4 hours, about 4 hours toabout 6 hours, about 6 hours to about 8 hours, or about 8 hours aftersampling. Rapid refers to a period less than about 10 min, about 10 minto about 9 min, about 9 min to about 8 min, about 8 min to about 7 min,7 min to about 6 min, about 6 min to about 5 min, about 5 min to about 4min, about 4 min to about 3 min, about 3 min to about 2 min, about 2 minto about 1 min, or about 1 min.

The method may comprise detergents. The detergents may comprise, but arenot limited to, other detergents, such as Triton X-100, Triton X-114,Igepal® CA-630, n-dodecyl-β-D-maltoside (DDM), beta-decyl-maltoside,octyl-glucoside, digitonin, polysorbate 20, polyoxyethylene sorbitanmonolaurate, PEG (20) sorbitan monolaurate, polysorbate 80,polyoxyethylene sorbitan monooleate, PEG (80) sorbitan monooleate,glyco-lithocholate amphiphiles (GLC-1, GLC-2, and GLC-3),glyco-diosgenin amphiphile (GDN),3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), or acombination thereof. The detergents may comprise non-ionic detergents.The non-ionic detergents may comprise uncharged hydrophilic headgroups.The non-ionic detergents may be mild surfactants capable of breakingprotein-lipid and lipid-lipid associations, but not protein-proteininteractions. The non-ionic detergents may not be capable of denaturingproteins. The method may comprise using a solvent for detergents. Thesolvent may comprise, but is not limited to, phosphate buffered saline(PBS), tris(hydroxymethyl)aminomethane (TRIS),(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES),2-ethanesulfonic acid (MES), any other biological buffer, anynon-biological buffer, or a combination thereof.

The method may comprise using inhibitors of phosphatases, kinases, orproteases. Phosphatases, kinases, proteases, and other enzymes presentin the lysate may alter the activity of the enzyme of interest and aninhibitor may be used to inhibit the activity of unwanted enzymeactivity. Inhibitors of phosphatases, kinases, or proteases may be addedto the lysis solution. Inhibitors may inhibit proteases, including, butnot limited to, trypsin, chymotrypsin, and elastase; cysteine proteases,including but not limited to, papain, calpain and lysosomal cathepsins;aspartic proteases, including but not limited to, pepsin and rennin; andmetallo-proteases, including but not limited to, thermolysin andcarboxypeptidase A. Inhibitors may be present as individual compounds oras mixtures.

The method may measure enzyme activity in host cells infected withpathogens that were able to evade the neutralizing activity of anantibody or other blocking agent. Cleavage may be monitored in intactcells or in lysates from cells that were pretreated with antibody andtransfected with the substrate. The biomarker within the method may bean enzyme, e.g., PLpro, that is produced only by live virus in infectedcells.

The method may be used in high-throughput screens. In high-throughputscreens, large numbers of single measurements of unknown samples arecompared to positive and negative control samples. Prior to starting alarge screen, smaller test screens are used to assess the quality of anassay. The Z′ factor is used as a statistical measure that predicts thesuitability of an assay for screening. Z′ takes into account 4parameters: the means (p) and the standard deviations (G) of thepositive and the negative control. The controls are equivalent to theupper and lower limit of the assay. The Z′ factor is calculatedaccording to the following equation:

$Z^{\prime} = {1 - {\frac{{3\sigma\max} - {3\sigma\min}}{{\mu\max} - {\mu\min}}.}}$

A Z′ of >0.5 indicates that an assay has good performance for drugscreening. The method may generate Z factors between approximately 0.7to 0.84 at various enzyme concentrations.

PLpro, is an essential enzyme that has multiple roles: it cleaves theviral polyprotein and has ubiquitin and interferon-stimulated gene 15(ISG15) cleaving activities. The multiple roles of PLpro and the highconservation of its active sites make PLpro an excellent therapeutictarget for small molecule libraries

Turning now to the drawings, FIG. 1 shows a schematic representation ofa quench mechanism based on electron transfer. A synthetic peptidecomprising four amino acids (circles) is labeled with the fluorophoreAMC. In the intact peptide, the fluorescence from the fluorophore isquenched by the amide bond between the terminal amino acid of thepeptide. When the peptide is cleaved by PLpro, one peptidic and onenon-peptidic fragment is produced. The emission is monitored at 465 nmwith excitation at 365 nm.

FIG. 2 is a progress curve showing a fluorescence increase throughcleavage of the electron transfer substrate from FIG. 1 in crude tonguescrape lysate to which recombinant PLpro was added. The control reactiondoes not contain recombinant PLpro.

FIG. 3 shows a schematic representation of a quench mechanism based onFoerster Resonance Energy Transfer (FRET). A peptide comprising sevenamino acids (circles) is labeled on one end with the fluorophorefluorescein isocyanate and on the other with a quencher dinitrophenyl(DNP). When the peptide is excited using a wavelength of 490 nm, energyis transferred from the acceptor (fluorescein) to the donor. Uponcleavage, the donor and acceptor move apart and emission from theacceptor can be monitored at 520 nm.

FIG. 4 is a progress curve showing fluorescence increase throughcleavage of the FRET substrate from FIG. 3 by endogenous enzymes inthree dilutions of crude lysate from tongue scrapes. FIG. 5 shows amultiplexed measurement of the peptides from FIG. 1 and FIG. 3 uponcleavage by endogenous ACE2 and recombinant PLpro. The fluorescence ismeasured in a battery-operated handheld fluorometer.

FIG. 6 shows a progress curve using the FRET substrate of FIG. 3 infresh crude lysate or in crude lysate that was stored at 4° C. for 2hours. FIG. 7 shows a schematic of the enzymatic test procedure. Atongue scrape specimen is lysed in a vial containing lysis buffer, andthe lysate transferred to a vial containing the reaction components in afreeze-dried form. The vial is inserted into the measurement port of afluorometer and the fluorescence increase measured. FIG. 8 shows acalibrator curve of serial dilutions of unconjugated7-amino-methylcoumarin (AMC).

FIG. 9 shows a schematic of a lateral flow assay based on the SARSprotease cleavage assay. Substrates are labeled with histidine (His-6)and biotin (black circle) and reacted with PLpro enzyme. A mixture ofcleavage products is generated containing uncleaved fragments or thepeptidic fragment and biotin. The mixture is added to the sample pad ofa lateral flow device and the fragments moved to the absorption pad bycapillary flow. Uncleaved fragments and free biotin are captured bystreptavidin on the strip. Cleaved fragments are captured by immobilizedanti-histidine antibodies and detected by labeled anti-substrateantibodies. FIG. 10 shows a progress curve using either peptide with theamino acid sequence L-R-G-G-AMC (SEQ ID NO. 5) or Tyr-Dap-G-G-AMC in thepresence (+) or absence (−) of crude tongue scrape lysate. The peptidewith the sequence Tyr-Dap-G-G is not cleaved by endogenous enzymespresent in lysate, whereas L-R-G-G (SEQ ID NO. 1) is cleaved. FIG. 11shows an enzyme concentration curve using recombinant SARS PLpro andISG15-AMC (circles) or L-R-G-G-AMC (SEQ ID NO. 5) (triangles). SARSPLpro cleaves the substrate ISG15, which contains the tetrapeptidecleavage sequence L-R-G-G (SEQ ID NO. 1) at its carboxyterminus, around105 times more efficiently than the tetrapeptide L-R-G-G (SEQ ID NO. 1)alone. FIG. 12 shows an enzyme concentration curve using recombinantNL62 PLP2 and Ubiquitin-AMC (circles), ISG15-AMC (squares), ortetrapeptide-AMC (triangles). NL63 PLP2 cleaved ubiquitin 2.5 times moreefficiently than ISG15 and 120 times more efficiently than thetetrapeptide.

FIG. 13 shows an in vivo experiment that compares ISG15-AMC cleavagebetween crude lysates from 5970 human cells infected with 15,000 229Ecoronaviruses and uninfected lysates. FIG. 14 shows a schematic showinga peptide combination that results in superior sensitivity conferred bythe ISG15 substrates and specificity conferred by the tetrapeptidesequence Tyr-Dap-G-G. The data in FIG. 13 show that coronavirus proteaseactivity was detectable in as few as 6,000 cells that were infected with15,000 229E coronaviruses. In clinical samples, it is expected to obtainaround 800,000 shed epithelial cells/test (100 μl) in saliva or fromtongue scrapes, of which 5-10% (40,000-80,000 cells) can be infected.This translated to an average of 10× more cells than are detectable inthe in vivo model test with 6,000 cells. By comparison, current antigentests required 2 million to 20 million viral copies for detection. Theenzymatic test is thus more than 300 times more sensitive than antigentests. RT-PCR tests require between 500-80,000 copies/ml while theenzymatic activity test is within an order of magnitude of thissensitivity, and given the advantages of this test, equals RT-PCR inclinical samples.

FIG. 15 shows an in vivo test using crude lysates from human cells thatwere infected with 229E, 0043 or NL63 coronaviruses. The cleavage ofsubstrates Tyr-Dap-G-G, L-R-G-G (SEQ ID NO. 1), mono-ubiquitin (UbiQ)and ISG15 was shown as the percent fluorescence increase relative touninfected cells. Cleavage was measured after 15 minutes (*), 60 minutes(**), and 800 minutes (***). In vivo studies on human coronavirusstrains 0043, 229E and NL63 with the enzymatic activity assay showedthat the strains have different preferences for various substrates thatcontain the cleavage site L-R-G-G (SEQ ID NO. 1) at the C-terminus.

FIG. 16 shows an in vivo test in which crude lysates from human cellsthat were infected with 15,000, 3,750, or 0 229E coronaviruses cleavethe ACE2-FITC substrate from FIG. 3 .

FIG. 17 shows a test in which florescence from eight replicates of wellscontaining the substrate L-R-G-G-AMC (SEQ ID NO. 5) and variousconcentrations of recombinant SARS PLpro enzyme were measured todetermine the Z′.

INDUSTRIAL APPLICABILITY

The invention is further illustrated by the following non-limitingexamples. The ubiquitin used in experiments was a monoubiquitin. Inaddition, the tests used, for example, a panel of synthetic orrecombinant ubiquitins that contain all eight native-linked diubiquitinconjugates: M1 (linear), K6, K11, K27, K29, K33, K48 and K63 linked.Other branched and linear ubiquitins are available. The preference forthe substrates was determined in a competitive assay using theubiquitins together with AMC or ACC-labeled tetrapeptide and recombinantPLpro or PLP enzyme from each coronavirus. The higher the fluorescencerecorded, the lower the preference for binding and cleaving theunlabeled ubiquitin. Alternatively, direct labeled substrates may beused and cleavage measured in a direct, non-competitive assay. Thisapproach delivers specific cleavage fingerprints of different humancoronaviruses, e.g., from HCoV SARS-1, SARS-2, MERS, 229E, NL64, HKU1,0043 that allow identifying the causative coronaviruses anddistinguishing respiratory infections caused by coronavirus from otherviruses that do not have delSGlating and deubiquitinylating activities,such as for example influenza, syncytial, parainfluenza viruses.

Example 1

Homogeneous SARS PLpro protease activity assay in crude lysate wasassessed. PLpro activity may be detected using peptide substrates basedon the amino acid sequence L-R-G-G (SEQ ID NO. 1) that is tagged withthe fluorophore 7-amino-methylcoumarin (AMC). When cleavage occurredbetween the terminal glycine and AMC, fluorescence was released and wasmonitored using excitation and emission wavelengths of 365 nm and 465nm, respectively.

Assays were performed in 7.5 μl volumes in 384-well plates at roomtemperature containing 50 μM substrate and recombinant SARS PLpro (100nM; BPS Biosciences) in 50 mM HEPES, pH 7.0, 5 mM DTT. Cells wereharvested from tongue scrapes and the cells lysed in ddH₂0 containingvarious detergents. 7.5 μl of the crude lysate was added to the reactionmixture in the wells and the reaction progress was monitored at roomtemperature in 1-minute intervals using a plate-based fluorometer(Molecular Devices). The increase in fluorescence was measured as afunction of enzyme activity. The control well did not containrecombinant enzyme. The slight fluorescence increases in the controlwell were due to cleavage of the substrate L-R-G-G-AMC (SEQ ID NO. 5) byendogenous enzymes present in the lysate. A substrate with artificialamino acids (Tyr-Dap-G-G) was not cleaved in crude human lysate.

Example 2

ACE2 was used as an internal control in crude lysate. An internalcontrol, Angiotensin-Converting Enzyme2, was used to confirm thatlysates were of sufficient quality and quantity for the assay to beperformed. Fluorescence increase was monitored using a FRET peptidesubstrate with the sequence FITC-C6-Tyr-Val-Ala-Asp-Ala-Pro-Lys(Dnp)-OH(SEQ ID NO. 6). Fluorescein isocyanate (FITC) was quenched bydinitrophenyl (DNP). The substrate (5 μM) was diluted in 2× assay buffer(50 mM HEPES, pH 7.0, 5 mM DTT) and crude lysate was added to obtain afinal volume of 15 μl in a well of a 384-well plate. The reactionprogress was monitored in 1-minute intervals using a plate-basedfluorometer with excitation and emission wavelengths of 490 nm and 515nm. It was observed that fluorescence increased as a function of thenumber of coronaviruses used to infect cells, indicating increased ACE2activity (or caspase activity) in cells with higher viral load.

Example 3

A multiplexed PLpro/ACE2 assay was performed. Reactions were performedas described in Example 1 and 2 in one vial and the fluorescence of eachfluorophore was measured in a volume of 80 μl. The solution was preparedin a 0.5 ml real time PCR tube which was inserted into abattery-operated fluorometer. The fluorescence from the reactions wasmeasured consecutively by switching from channel 1 (λexc=365 nm/λem=465nm) to channel 2 (λexc=490 nm/λem=520 nm) by the push of a button.

Example 4

Lysate stability was measured. The activity of crude lysate from atongue scrape sample stored for 2 hours on ice was compared to crudefresh lysate from the same individual. The fluorescence from the ACE2reactions was measured in 1-minute intervals using λexc=490 nm andλem=520 nm in a plate-based fluorometer. Lysate without substrate andsubstrate without lysate were used as controls.

Example 5

Viral load was quantitatively measured. The fluorescence fromunconjugated fluorophore, for example 7-amino-methycoumarin (AMC), wasused to calculate the number of viruses that are present in a crudelysate that tested positive for PLpro activity. To this end, acalibrator curve was prepared containing several dilutions of AMC andthe fluorescence was compared to the fluorescence obtained from a serialdilution of PLpro. To normalize for the amount of lysate present, aprotein binding or DNA intercalating dye was used, such as Quantifluoror Hoechst 33258.

The preceding examples can be repeated with similar success bysubstituting the generically or specifically described components and/oroperating conditions of embodiments of the present invention for thoseused in the preceding examples.

Note that in the specification and claims, “about” or “approximately”means within twenty percent (20%) of the amount or value given.

Embodiments of the present invention can include every combination offeatures that are disclosed herein independently from each other.Although the invention has been described in detail with particularreference to the disclosed embodiments, other embodiments can achievethe same results. Variations and modifications of the present inventionwill be obvious to those skilled in the art and it is intended to coverin the appended claims all such modifications and equivalents. Theentire disclosures of all references, applications, patents, andpublications cited above are hereby incorporated by reference. Unlessspecifically stated as being “essential” above, none of the variouscomponents or the interrelationship thereof are essential to theoperation of the invention. Rather, desirable results can be achieved bysubstituting various components and/or reconfiguring their relationshipswith one another.

What is claimed is:
 1. A method for detecting a coronavirus, the methodcomprising: providing a specimen, wherein the specimen comprises aplurality of cells; contacting the specimen with a first substrate,wherein the first substrate comprises a first signaling moietyconjugated to a carboxy terminus of the first substrate, the firstsubstrate further comprising an Interferon Stimulated Gene-15 (ISG-15)or a fragment thereof, a ubiquitin or a fragment thereof, or acoronavirus protein fragment, and wherein the first substrate is cleavedby a coronavirus enzyme; contacting the specimen with a secondsubstrate, wherein the second substrate comprises a control peptide anda second signaling moiety conjugated to the control peptide, wherein thesecond substrate is cleaved by an internal control enzyme; determiningthe presence of a first fluorescent signal from an unconjugated firstsignaling moiety produced by the coronavirus enzyme cleaving the firstsubstrate wherein the presence of the first fluorescent signal indicatesa presence of the coronavirus in the specimen; determining the presenceof a second fluorescent signal from an unconjugated second signalingmoiety produced by the internal control enzyme cleaving the controlpeptide wherein the presence of the second fluorescent signal indicatesa presence of the internal control enzyme within the cell; anddetermining the first fluorescent signal from the unconjugated firstsignaling moiety and the second fluorescent signal from the unconjugatedsecond signaling moiety to detect the coronavirus, the internal controlenzyme or both in the specimen.
 2. The method of claim 1, wherein thefirst substrate is added directly to live cells within the specimen. 3.The method of claim 1, further comprising lysing the plurality of cellsprior to contacting the specimen with the first substrate.
 4. The methodof claim 1, wherein the coronavirus is severe acute respiratory syndromecoronavirus 2 (SARS-CoV-2).
 5. The method of claim 1, wherein theinternal control enzyme is angiotensin-converting-enzyme (ACE2).
 6. Themethod of claim 1, wherein the coronavirus enzyme is papain-likeprotease (PLpro).
 7. The method of claim 1, further comprisingquantifying an amount of the coronavirus present in the specimen bycomparing the first fluorescent signal to a calibration curve ofrelative fluorescence versus a fluorophore concentration.
 8. The methodof claim 1, wherein the first signaling moiety comprises rhodamine 110and wherein the determining the presence of the first fluorescent signalcomprises exciting the rhodamine 110 with a first wavelength of lightand detecting the first fluorescent signal at a second wavelength oflight different from the first wavelength of light.
 9. The method ofclaim 1, wherein the first substrate comprises ISG-15 or an ISG-15fragment and a ubiquitin or a ubiquitin fragment.
 10. The method ofclaim 1, wherein the ISG-15 fragment comprises a sequence selected from(SEQ ID NO. 1), (SEQ ID NO. 4), (SEQ ID NO. 5), (SEQ ID NO. 6), (SEQ IDNO. 7), (SEQ ID NO. 8), (SEQ ID NO. 9), (SEQ ID NO. 10), (SEQ ID NO.11), (SEQ ID NO. 12) and (SEQ ID NO. 13).
 11. The method of claim 1,wherein the specimen is provided in a plurality of wells within amulti-well plate and the multi-well plate comprises a calibrator withinat least one well.
 12. The method of claim 1, wherein the specimen isprovided in a plurality of wells within a multi-well plate and furthercomprising quantifying the coronavirus load within a well.
 13. Themethod of claim 1, wherein the specimen is provided in a plurality ofwells within a multi-well plate and the plate includes a positivecontrol and a negative control.
 14. The method of claim 1, wherein thespecimen is provided in a plurality of wells within a multi-well plateand further comprising treating the specimen with an antibody andsubsequently transfecting the plurality of cells with a substrate. 15.The method of claim 1, wherein the specimen is provided in a pluralityof wells within a multi-well plate and further comprising treating thespecimen with an antibody and subsequently transfecting the plurality ofcells with the first substrate.
 16. The method of claim 1, wherein thespecimen is provided in a plurality of wells within a multi-well plateand further comprising treating the specimen with an antibody andsubsequently transfecting the plurality of cells with the secondsubstrate.
 17. The method of claim 1, wherein the specimen is providedin a plurality of wells within a multi-well plate and further comprisingtreating the specimen with an inhibitor.
 18. The method of claim 1further comprising differentiating between a first coronaviruscomprising a first coronavirus enzyme and a second coronaviruscomprising a second coronavirus enzyme by comparing the firstfluorescent signal from the unconjugated first signaling moiety cleavedfrom the first substrate by the first coronavirus enzyme over a periodof time with the first fluorescent signal from the unconjugated firstsignaling moiety cleaved from the first substrate by the secondcoronavirus enzyme over the period of time.
 19. The method of claim 1further comprising deriving the first substrate from a selected one ofat least two different species to identify the coronavirus.
 20. Themethod of claim 1, wherein the first substrate further comprises aquencher comprising an organic dye or a molecule with an amide bond, andwherein the first signaling moiety is a fluorophore comprising xanthenedyes, coumarins, benzamide dyes, phenathridine dyes, acridine dyes,cyanine dyes, carbazole dyes, phenoxazine dyes, porphorin dyes,quinoline dyes, or a combination thereof.
 21. The method of claim 1,wherein the first substrate further comprises a quencher comprising anorganic dye or a molecule with an amide bond, and wherein the firstsignaling moiety is a fluorophore comprising aminomethyl coumarin (AMC),aminocarbomylcoumarin (ACC), rhodamine 110, or a combination thereof.22. The method of claim 1, wherein the second substrate furthercomprises a quencher comprising an organic dye or a molecule with anamide bond, and wherein the second signaling moiety is a fluorophorecomprising xanthene dyes, coumarins, benzamide dyes, phenathridine dyes,acridine dyes, cyanine dyes, carbazole dyes, phenoxazine dyes, porphorindyes, quinoline dyes, or a combination thereof.
 21. The method of claim1, wherein the second substrate further comprises a quencher comprisingan organic dye or a molecule with an amide bond, and wherein the secondsignaling moiety is a fluorophore comprising aminomethyl coumarin (AMC),aminocarbomylcoumarin (ACC), rhodamine 110, or a combination thereof.